1. Field of the Invention
This invention relates to improved vectors and methods for producing polypeptides using such vectors. In particular, this invention is related to improved expression of polypeptides from nucleic acids such as cloned genes and production of various polypeptides and proteins, including those of eukaryotic origin in prokaryotic hosts.
2. Description of Related Art
The level of production of a protein in a host cell is governed by three major factors: the number of copies of its gene within the cell, the efficiency with which those gene copies are transcribed, and the efficiency with which the resultant messenger RNA (mRNA) is translated. The quality of protein produced is similarly governed by various factors, including the anti-termination mechanism in the host cell.
Recombinant proteins produced in E. coli occasionally contain structural modifications that restrict their usefulness as therapeutic drugs or reagents for structure-function relationship studies. Such modifications include N- and C-terminal truncations, extensions, incomplete removal of N-terminal initiator methionine, misincorporation of lysine for arginine, and norleucine for methionine. For example, during the purification of recombinant murine interleukin-6 from E. coli, it was observed that 5–10% of the mIL-6 molecules contained a novel C-terminal modification (Tu et al., J. Biol. Chem., 270: 9322–9326 (1995)).
This C-terminal “tag” is encoded by a small metabolically stable RNA of E. coli (10Sa RNA) (Chauhan and Apirion, Mol. Microbiol., 3: 1481–1485 (1989)). 10Sa RNA, also known as transfer-messenger RNA, or tmRNA, contains a tRNA-like structure in vivo with the 5′- and 3′-end sequences and an internal reading frame encoding a “tag” peptide.
The primary cause of the production of truncated 10Sa-tagged proteins is the translation of mRNA truncated within the coding region (Keiler et al., Science, 271: 990–993 (1996)). Premature transcription termination and RNase cleavage appear to be the major factors capable of producing such truncated mRNA. The first of these factors, premature transcription termination, is potentially amenable to some type of transcription anti-termination. Several of these systems have been described, including λN, λQ, HK022, rrn, and Psu (Weisberg et al., J. Bacteriol., 181: 359–367 (1999)). Most of these systems are used to control gene expression temporally in phage development by transcribing through intergenic transcription terminators. The function of the rrn anti-termination is somewhat different, and it has been proposed to prevent rho-dependent transcription termination within the non-translated ribosomal RNA operons.
Despite the accumulation of considerable knowledge of these systems over many years, their only demonstrated usefulness in terms of recombinant technology has been in the control of gene expression by overriding intergenic transcriptional terminators (Mertens et al., Bio/Technol., 13: 175–179 (1995)). Other reports describe the failure of one of these systems (rrn) to alleviate problems within a translated coding sequence containing extensive secondary structure (Makrides, Microbiol. Rev., 60: 512–538 (1996)).
Several fusions of a protein with at least a portion of an anti-terminator protein have been disclosed, especially the N gene protein and most particularly the N-terminal fragment thereof (JP 9059299 published Mar. 4, 1997; WO 89/03886 published May 5, 1989; WO 88/06628 published Sep. 7, 1988; U.S. Pat. No. 5,834,184 issued Nov. 10, 1998; EP 700,997 published Mar. 13, 1996; U.S. Pat. No. 5,354,846 issued Oct. 11, 1994; U.S. Pat. No. 5,618,715 issued Apr. 8, 1997; U.S. Pat. No. 5,374,520 issued Dec. 20, 1994; Zhukovskaya et al., Nucl. Acids. Res., 20: 6081–6090 (1992); Horiuchi et al., Biotechnol. Lett., 16: 113–118 (1994); Kamasawa et al., IFAC Symp. Ser., 10: 255–258 (1992); Kovgan et al., Vopr. Virusol., 31: 485–489 (1986)).
Several plasmids that contain an N utilization site for binding anti-terminator N protein produced by the host cell, such as E. coli, have been constructed (U.S. Pat. No. 5,256,546 issued Oct. 26, 1993; EP 691,406 published Jan. 10, 1996; U.S. Pat. No. 5,162,217 issued Nov. 10, 1992; EP 131,843 published Jan. 23, 1985). Other plasmids involving the N gene, operon, or portion thereof have been described (SU 1405313 published Mar. 15, 1994; EP 314,184 published May 3, 1989; U.S. Pat. No. 4,578,355 issued Mar. 25, 1986; WO 85/04418 published Oct. 10, 1985; Rees et al., Proc. Natl. Acad. Sci. USA, 93: 342–346 (1996); Hwang et al., Biochem. Biophys. Res. Commun., 173: 711–717 (1990); Bielawski et al., Acta Biochim. Pol., 34: 29–34 (1987); Stanssens et al., Cell, 44: 711–718 (1986); Gatenby and Castleton, Mol. Gen. Genet., 185: 424–429 (1982)); Martin-Gallardo et al., J. Gen. Virol., 74: 453–458 (1993); Das, 72nd Annual Meeting of the American Society of Biological Chemists, May 31–Jun. 4, 1981, Fed. Proc., 40 (6): 1764 (1981); Beck et al., Bio/Technology, 6: 930–935 (1988)).
The expression of gamma-interferon was found to increase over two-fold when the λN anti-termination system was eliminated and only the PL promoter was used (WO 85/02624 published Jun. 20, 1985). Cloning and expression vectors in which the active N gene is preferably absent are also described (U.S. Pat. No. 5,401,658 issued Mar. 28, 1995).
Transcription of DNA is often arrested at sites in DNA that trap a fraction of elongating RNA polymerase molecules that pass through, resulting in locked ternary complexes that cannot propagate or dissociate their RNA product. Transcript cleavage factors cleave the RNA in such complexes at the 3′ end, allowing RNA polymerase to back up and re-attempt to read through the potential trap. In addition to assuring efficient transcript elongation, transcript cleavage factors increase the fidelity of transcription, since misincorporated bases at the 3′ end of the nascent RNA also lead to arrested complexes (Erie et al., Science, 262: 867–873 (1993)). Further, such factors allow RNA polymerase to transcribe through strong blocks to elongation that can otherwise arrest the enzyme on the DNA (Lee et al., J. Biol. Chem., 269: 22295–22303 (1994)). In addition, these factors can facilitate the transition of RNA from the stage of abortive initiation to elongation at certain promoters (Hsu et al., Proc. Natl. Acad. Sci. USA, 92: 11588–11592 (1995)).
Both bacteria and eukaryotes contain proteins that can stimulate such cleavage (Surratt et al., Proc. Natl. Acad. Sci. USA, 88: 7983–7987 (1991); Borukhov et al., Proc. Natl. Acad. Sci. USA, 89: 8899–8902 (1992); Borukhov et al., Cell, 72: 459–466 (1993); Izban and Luse, Genes & Dev., 6: 1342–1356 (1992); Izban and Luse, J. Biol. Chem., 267: 13647–13655 (1992); Izban and Luse, J. Biol. Chem., 268: 12864–12873 (1993); Izban and Luse, J. Biol. Chem., 268: 12874–12885 (1993); Kassavetis and Geiduschek, Science, 259: 944–945 (1993); Reines, J. Biol. Chem., 267: 3795–3800 (1992); Wang and Hawley, Proc. Natl. Acad. Sci. USA, 90: 843–847 (1993); Gu et al., J. Biol. Chem., 268: 25604–25616 (1993); Guo and Price, J. Biol. Chem., 268: 18762–18770 (1993)). Two modes of cleavage have been described. One yields one to three nucleotide fragments and the other produces larger fragments, up to at least 12 nucleotides in size. Two transcript cleavage factors, GreA and GreB, have been identified in E. coli (Borukhov et al., Proc. Natl. Acad. Sci. USA, supra, and Borukhov et al., Cell, supra, respectively). GreA-dependent transcript cleavage usually results in the removal of di- and trinucleotides from the 3′ end of the stalled RNA. GreB-dependent cleavage yields larger oligonucleotides, up to a length of nine nucleotides. Both proteins bind RNA polymerase. Neither the GreA nor GreB proteins possess intrinsic nuclease activity; rather, they stimulate a nuclease activity inherent in RNA polymerase (Oriova et al., Proc. Natl. Acad. Sci. USA, 92: 4596–4600 (1995)). The GreA and GreB proteins are homologous, sharing 38% sequence identity and 59% sequence similarity. It was found that GreA-induced transcript cleavage in transcription complexes containing E. coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure (Feng et al., J. Biol. Chem., 269: 22282–22294 (1994)).
Crystallization of GreA has been disclosed (Darst et al., J. Mol. Biol., 242: 582–585 (1994)) as well as its crystal structure (Stebbins et al., Nature, 373: 636–640 (1995)). The organization and functions of domains of GreA and/or GreB have been investigated (Koulich et al., J. Biol. Chem., 272: 7201–7210 (1997); Koulich et al., J. Mol. Biol., 276: 379–389 (1998); Polyakov et al., J. Mol. Biol., 281: 465–473 (1998)). Moreover, purification and assay procedures for GreA and GreB are reported (Borukhov and Goldfarb, Meth. Enzymol., 274: 315–326 (1996)). Interactions between RNA polymerase and transcript affect GreA- and GreB-mediated reverse translocation (Feng et al., J. Cellular Biochem. Suppl., 0: 18C, p. 58 (1994)). Both GreA and GreB have been shown to enhance promoter escape (Hsu et al., Proc. Natl. Acad. Sci. USA, 92: 11588–11592 (1995)).
In eukaryotes, the transcription elongation factor TFIIS, otherwise known as SII (Reines et al., J. Biol. Chem., 264: 10799–10809 (1989); Sluder et al., J. Biol. Chem., 264: 8963–8969 (1989)), is similar to the GreA and GreB proteins in that it stimulates RNA cleavage from the 3′ end of RNA in a stalled complex but does not share significant sequence homology with the GreA and GreB proteins (Borukhov et al., Cell, supra). TFIIS stimulates either small- or large-fragment cleavage, depending on reaction conditions and the particular complex examined (Izban and Luse, J. Biol. Chem., 268: 12874–12885 (1993), supra; Wang and Hawley, supra). Evidence for functional similarity between prokaryotic and eukaryotic transcription elongation and read-through mechanisms has been found (Mote and Reines, J. Biol. Chem., 273: 16843–16852 (1998)).
Homologs of E. coli GreA have been identified. The predicted amino acid sequence encoded by the Rickettsia prowazekii greA gene has 50.3% amino acid identity and 66.9% amino acid similarity to E. coli GreA (Marks and Wood, Nucl. Acids Res., 20: 3785 (1992)). The deduced amino acid sequence of GreA from Pseudomonas aeruginosa exhibits 65.2% identity to its counterpart in E. coli K-12 (Lu et al., J. Bacteriol., 179: 3043–3046 (1997)). Streptococcus pneumoniae polypeptide GreA has also been disclosed, along with methods of producing the GreA polypeptide by recombinant means and for utilizing GreA or its antagonists for the treatment or diagnosis of infection (EP 838,525 published Apr. 2, 1998). Further, GreA from Staphylococcus aureus has also been disclosed, as well as recombinant methods of making it and methods for utilizing it to screen for antibacterial compounds (EP 893,502 published Jan. 27, 1999).
There is a current and continuing need in the art for improving the quality of recombinant protein produced by host cells such that production of truncated forms of the protein, such as 10Sa-tagged material, is minimized or eliminated. There is also a need for higher amounts of full-length protein produced by prokaryotes.